阅读说明：本技术 靶向肿瘤周围细胞激活免疫的聚合物及其制备方法与应用 (Polymer for activating immunity by targeting tumor peripheral cells and preparation method and application thereof ) 是由 帅心涛 林敏钊 蔡宇骏 陈耿佳 于 2021-07-06 设计创作，主要内容包括：本发明属于纳米医学与生物医学工程技术领域,具体涉及一种靶向肿瘤周围细胞激活免疫的聚合物及其制备方法与应用。该聚合物由PD-L1抗体-基质金属蛋白酶蛋白2-聚乙二醇-聚乙烯亚胺和甘露糖-聚乙二醇-聚乙烯亚胺作为嵌段聚合物载体,复合上dsDNA制成,以PD-L1抗体靶向,提高纳米聚合物的聚集、增强抗体滞留的同时解除肿瘤周围免疫细胞的免疫阻断；协同激活肿瘤周围免疫细胞的STING通路改善肿瘤微环境募集T细胞,而不是直接作用于肿瘤细胞,通过“旁观者”效应实现杀伤周围肿瘤细胞的效果,应用前景广阔。(The invention belongs to the technical field of nano-medicine and biomedical engineering, and particularly relates to a polymer for activating immunity by targeting tumor peripheral cells, and a preparation method and application thereof. The polymer is prepared by compounding a PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine and mannose-polyethylene glycol-polyethyleneimine as block polymer carriers with dsDNA, and is targeted by a PD-L1 antibody, so that the aggregation of a nano polymer is improved, the retention of the antibody is enhanced, and the immune block of immune cells around a tumor is relieved; the STING passage synergistically activating the immune cells around the tumor improves the tumor microenvironment to recruit T cells instead of directly acting on the tumor cells, realizes the effect of killing the surrounding tumor cells through the 'bystander' effect, and has wide application prospect.)

1. A polymer for activating immunity by targeting cells around tumors is characterized in that the polymer is prepared by compounding dsDNA (deoxyribonucleic acid) on a block polymer carrier which is PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine and mannose-polyethylene glycol-polyethyleneimine.

2. The polymer for targeting peripheral tumor cell activation immunity according to claim 1, wherein the dsDNA is double-stranded by the complexation of two DNA oligos, wherein the sequences of the two DNA oligos are 5'-TACAGATCTACTAGTGATCTATG-3', 5'-ACTGATCTGTACATGATCTACA-3' respectively.

3. The polymer for activating immunity by targeting cells around tumor according to claim 1, wherein the molecular weight of the polyethylene glycol is 1000-2000 Da.

4. The polymer for targeting peripheral tumor cells and activating immunity according to claim 1, wherein the molecular weight of the polyethyleneimine is 600-1800 Da.

5. The polymer for targeting peripheral tumor cell activation immunity according to claim 1, wherein the molar ratio of nitrogen to phosphorus in dsDNA of the polymer is 10-40.

6. The method for preparing the polymer for activating immunity by targeting the cells around the tumor according to any one of claims 1 to 5, which comprises the following steps:

s1, preparation of mannose-polyethylene glycol-polyethyleneimine: compounding mannose groups to a polyethylene glycol section on a block polymer Boc-PEG2000-NHS through amine reaction, adding (S) -4-benzyl-2-oxazolidone for ring-opening polymerization after removing Boc, and adding polyethyleneimine for full reaction to obtain mannose-polyethylene glycol-polyethyleneimine;

preparation of S2, PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine: n is a radical of₃-PEG2000-NH₂Performing ring-opening polymerization reaction with (S) -4-benzyl-2-oxazolidinone, adding polyethyleneimine to perform full reaction, adding MMP2 to enable sensitive peptide Mal groups to perform click reaction with azide groups of polyethylene glycol, and finally adding aPD-L1 to react with the sensitive peptide Mal groups through surface sulfhydryl groups to obtain PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine;

s3, preparation of polymer: and (3) compounding the mannose-polyethylene glycol-polyethyleneimine obtained in the step S1 and the PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine obtained in the step S2 with dsDNA to form a polymer.

7. The method of claim 6, wherein the sensitive peptide Mal group of MMP2 has a structure of Mal-GPLGVRG-Pra in step S2.

8. The method of claim 6, wherein in step S3, the polyethyleneimine at one end of mannose-polyethylene glycol-polyethyleneimine and PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine is complexed with the negatively charged dsDNA by positive and negative electro-adsorption of the polyamino structure.

9. Use of the polymer for targeting tumor-surrounding cell-activated immunity according to any one of claims 1 to 6 in the preparation of a medicament for treating cancer.

10. The use of claim 9, wherein the cancer is breast, pancreatic, prostate, or liver cancer.

Technical Field

The invention belongs to the technical field of nano-medicine and biomedical engineering. More particularly, relates to a polymer for activating immunity by targeting cells around tumors, and a preparation method and application thereof.

Background

Immune Checkpoint Blockade (ICB) has become one of the most attractive and effective means in cancer immunotherapy. In particular, blockade of the programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) axis, can stress depleted T cells to inhibit tumor growth. However, in practical clinical applications, more than half of patients fail to benefit from the PD-1/PD-L1 blockade due to the multiple immune evasion mechanisms developed by tumors, and the therapeutic effect is limited.

The combination of immune checkpoint blockade with immunomodulators and the like to improve the tumor microenvironment has become a research hotspot in cancer treatment. For example, activation of the innate immune pathway of interferon gene Stimulation (STING) has become an effective strategy for cancer treatment, which can promote secretion of type I interferon (IFN-I) and production of various pro-inflammatory and chemotactic factors, and induce activation of the adaptive immune system through a series of cascade reactions, ultimately promoting proliferation and activation of effector T cells. Intratumoral (IT) injections of STING agonists have shown efficacy in mouse models and phase I clinical trials have begun (clinical trials. gov, NCT02675439 and NCT 03172936). In the last five years, companies such as Aduro/Nowa, Muscont, Merck, Kurarin Schke and the like successively develop small molecule agonists taking STING as a target point, and the small molecule agonists enter the clinical stage to be used for treating solid tumors and lymphomas singly or in combination, but the early clinical data of the products are disappointing, and the response rate of patients is not high. The rapid metabolism of small molecule drugs in vivo and poor selectivity to tumors become major obstacles restricting the safe and effective application of STING agonists in clinic.

Therefore, an effective strategy to deliver PD-L1 antibody (aPD-L1) or STING agonists to target cells with high efficiency is crucial for combination cancer therapy. In order to solve the above problems, the prior art considers that aPD-L1 and STING agonist are loaded on a specific carrier, as chinese patent application CN112601554A discloses a drug-binding agent conjugate activated by tumor microenvironment, which can be made of checkpoint protein and checkpoint antagonist PD-L1 and immunomodulator STING agonist, etc. for treating tumor; however, in practical application, the conjugate is difficult to deliver the PD-L1 and the STING agonist to proper positions respectively, and has the problems of difficult delivery, uneven distribution and the like; and when using nanocarriers for tumor-targeted drug delivery, solid tumors in the body may exhibit Enhanced Permeation and Retention (EPR) effects on large and nano-sized objects, resulting in passive tumor accumulation of the nanocarriers that is generally far from expected, with very limited tumor treatment effects. Therefore, it is highly desirable to provide a polymer that can overcome the tumor penetration and retention effects and achieve a targeted delivery that allows the drug to fully function.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects and shortcomings of the existing micromolecule drug such as fast metabolism, poor tumor selectivity, tumor penetration and retention effect and the like which are influenced by escape mechanisms, and provides a polymer capable of targeting cells around tumors.

The invention aims to provide a polymer which targets cells around tumors to activate immunity.

The invention also aims to provide a preparation method of the polymer for targeting the cells around the tumor to activate immunity.

The invention also aims to provide application of the polymer for targeting tumor surrounding cells to activate immunity in preparing a medicament for treating cancer.

The above purpose of the invention is realized by the following technical scheme:

a polymer for activating immunity by targeting cells around tumors is prepared by compounding dsDNA (deoxyribose nucleic acid) on a block polymer carrier which is PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine and mannose-polyethylene glycol-polyethyleneimine.

The polymer for activating immunity by targeting tumor peripheral cells takes a PD-L1 antibody on the surface of the polymer as a target head, and after the target is taken for the tumor peripheral cells, the abundant matrix metalloproteinase protein 2(MMP-2) in the tumor microenvironment breaks the antibody of the polymer, so that the specific blocking of the immune tolerance of the tumor is realized; meanwhile, the PD-L1 antibody targets immunosuppressive cells of high-expression PD-L1 protein around the tumor, and the effect of improving the aggregation capability of the nano polymer is achieved. In addition, the exposed mannose group as the outer end of the polymer can target immune cells (such as dendritic cells, macrophages and the like) and promote the immune cells to take up the polymer, and in the polymer taken up by the immune cells, the positive charge on the surface of polyethyleneimine is compounded with dsDNA capable of activating a STING pathway, so that the in vivo immune effect is activated, and T cell infiltration is promoted. In general, the polymer is targeted by a PD-L1 antibody, so that the aggregation of the nano polymer is improved, the retention of the antibody is enhanced, and the immune block of immune cells around the tumor is relieved; the STING passage synergistically activating the immune cells around the tumor improves the tumor microenvironment to recruit T cells instead of directly acting on the tumor cells, realizes the effect of killing the surrounding tumor cells through the 'bystander' effect, and has wide application prospect.

Further, the dsDNA is double-stranded by the complexing of two DNA oligos, wherein the sequences of the two DNA oligos are 5'-TACAGATCTACTAGTGATCTATG-3', 5'-ACTGATCTGTA CATGATCTACA-3', respectively. The cells recognize double-stranded DNA in cytoplasm through an upstream cGAS passage, and the double-stranded DNA is used as a danger signal to activate body immunity, so that secretion of type I interferon (IFN-I) and generation of various proinflammatory factors and chemotactic factors are promoted, activation of an adaptive immune system is induced through a series of cascade reactions, and finally proliferation and activation of effector T cells are promoted.

Furthermore, the molecular weight of the polyethylene glycol is 1000-2000 Da.

Furthermore, the molecular weight of the polyethyleneimine is 600-1800 Da.

Furthermore, the molar ratio of nitrogen in the polymer to phosphorus in the dsDNA is 10-40. Preferably, the molar ratio of nitrogen in the polymer to phosphorus in the dsDNA is 20-40.

In addition, the invention also provides a preparation method of the polymer for targeting the peripheral cells of the tumor to activate immunity, which comprises the following steps:

s1, preparation of mannose-polyethylene glycol-polyethyleneimine: compounding mannose groups to a polyethylene glycol section on a block polymer Boc-PEG2000-NHS through amine reaction, adding (S) -4-benzyl-2-oxazolidone for ring-opening polymerization after removing Boc, and adding polyethyleneimine for full reaction to obtain mannose-polyethylene glycol-polyethyleneimine;

preparation of S2, PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine: n is a radical of₃-PEG2000-NH₂Performing ring-opening polymerization reaction with (S) -4-benzyl-2-oxazolidinone, adding polyethyleneimine to perform full reaction, adding MMP2 to enable sensitive peptide Mal groups to perform click reaction with azide groups of polyethylene glycol, and finally adding aPD-L1 to react with the sensitive peptide Mal groups through surface sulfhydryl groups to obtain PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine;

s3, preparation of polymer: and (3) compounding the mannose-polyethylene glycol-polyethyleneimine obtained in the step S1 and the PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine obtained in the step S2 with dsDNA to form a polymer.

Further, in step S2, the sensitive peptide Mal group structure of MMP2 is Mal-GPLGVRG-Pra.

Further, in step S3, the polyethyleneimine at one end of the mannose-polyethylene glycol-polyethyleneimine and PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine is complexed with the negatively charged dsDNA by the over-positive and negative electro-adsorption of the polyamino structure.

In addition, the invention also provides application of the polymer for targeting tumor surrounding cells to activate immunity in preparing a medicament for treating cancer. Based on the similar characteristics of cell compositions in a tumor microenvironment and the targeting and action principles of the polymer for targeting tumor surrounding cell activated immunity, the polymer for targeting tumor surrounding cell activated immunity is suitable for common solid tumors and can achieve the same effect.

Preferably, the cancer is breast cancer, pancreatic cancer, prostate cancer or liver cancer.

The invention has the following beneficial effects:

the polymer for activating immunity by targeting tumor peripheral cells adopts PD-L1 antibody targeting, improves the aggregation of the nano polymer, enhances the retention of the antibody and simultaneously relieves the immune block of the tumor peripheral immune cells; the STING passage synergistically activating the immune cells around the tumor improves the tumor microenvironment to recruit T cells instead of directly acting on the tumor cells, realizes the effect of killing the surrounding tumor cells through the 'bystander' effect, and has wide application prospect.

Drawings

FIG. 1 is a scheme showing the synthesis of mannose-polyethylene glycol-polyethyleneimine in example 1 of the present invention.

FIG. 2 is a synthetic scheme of PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine in example 1 of the present invention.

FIG. 3a is a TEM image of a polymer of the invention targeting tumor-surrounding cell activation immunity; FIGS. 3b, 3c are a bar graph and data statistics of the effect of dsDNA loading of the polymers of the invention targeting tumor-surrounding cell-activated immunity; FIG. 3d is a particle size histogram of the inventive polymer targeting tumor-surrounding cells to activate immunity.

FIG. 4 is a graph showing the nanoparticle uptake status of Raw264.7 cells in example 3.

FIG. 5 is an electrophoretogram of the cell protein immunoblotting experiment for verifying the STING activation status in example 4.

FIG. 6 is a graph showing the effect of in vivo aggregation of nanoparticles in example 5.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 preparation of Block Polymer support

1. Preparation of Mannose-polyethylene glycol-polyethyleneimine (Mannose-PEG 2000-PASp (-PEI))

The block polymer Boc-PEG_2K-NHS (0.1mmol), D-mannosamine hydrochloride (0.1mmol) dissolved in 10mL of N, N-dimethylformamide with 1,1,3, 3-tetramethylguanidine as protecting agent, reacted at 50 ℃ for 48 h; dialyzing with ultrapure water for 24h to obtain a product Boc-PEG2000-Mannose, and then reacting with trifluoroacetic acid/dichloromethane (1: 1) for 1h to remove Boc groups to obtain a product NH₂-PEG-manose; dissolving the obtained product in chloroform in an anhydrous environment, adding (S) -4-benzyl-2-oxazolidinone, and carrying out ring-opening polymerization for 48h to obtain a product, namely Mannose-PEG2000-PBLA, wherein the polymerization degree is 60; dissolving the product in dimethyl sulfoxide, adding 1.5-2 times of PEI (polyetherimide) in molar ratio, reacting overnight at 35 ℃, and dialyzing for 12 hours by methanol and water in sequence to obtain the product; the specific synthetic route is shown in figure 1.

2. Preparation of PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine [ aPD-L1-MMP sensitive peptide-PEG 2000-PASp (-PEI) ]

Will N₃-PEG2000-NH₂Dissolving the mixture in chloroform in an anhydrous environment, adding (S) -4-benzyl-2-oxazolidinone, and carrying out ring-opening polymerization for 48 hours to obtain a product N₃PEG2000-PBLA, here with a degree of polymerization of 60; dissolving the obtained product in dimethyl sulfoxide, adding 1.5-2 times of PEI (polyetherimide) in molar ratio, reacting overnight at 35 ℃, and dialyzing for 12 hours by methanol and water sequentially; then making the Mal group of MMP and azide group of polyethylene glycol produce click reaction, where the MMP2 sensitive peptide has the structure of Mal-GPLGVRG-Pra, finally aPD-L1 makes the surface sulfhydryl react with the Mal group on MMP sensitive peptide-PEG 2000-PASp (-PEI) to produce aPD-L1-MMP sensitive peptide-PEG 2000-PASp (-PEI); the specific synthetic route is shown in figure 2.

Example 2 preparation and characterization of polymers for targeting peritumoral cells to activate immunity

1. Preparation of polymer for activating immunity by targeting tumor peripheral cells

Taking mannose-polyethylene glycol-polyethyleneimine prepared in example 1 and PD-L1 antibody-matrix metalloproteinase protein 2-polyethylene glycol-polyethyleneimine, and forming nano polymer particles by compounding dsDNA.

Wherein the dsDNA is formed by the complex of two DNA oligos into a double chain, and the sequence structure is as follows: 5'-TACAGATCTACTAGTGATCTATG-3', 5'-ACTGATCTGTACATGATCTACA-3', respectively; the annealing steps are shown in table 1.

TABLE 1 annealing reagents and procedures

And (3) sequentially adding various reagents according to the sequence in the table 1, uniformly mixing, and setting a PCR instrument according to the steps 1-3 to perform annealing reaction to obtain the reagent.

2. Characterization of polymers targeting tumor-surrounding cell-activated immunity

The formation of dsDNA and the capability of loading dsDNA by nano polymer are verified by gel block electrophoresis experiment, which comprises the following steps:

(1) confirmation of dsDNA formation: preparing 2% agarose solution, pouring into a flat plate, adding 1.5 mu L nucleic acid dye, stirring uniformly, adding the same amount of dsDNA into each hole, adding 6X DNA loading into each sample, mixing uniformly, adding into a sample loading hole, performing electrophoresis at constant voltage of 100V for 30min, stopping an electrophoresis apparatus, taking out gel, placing the gel in a gel imaging system, observing an electrophoresis strip under an ultraviolet lamp, and taking a picture; referring to FIG. 3d, it can be seen that the DNA oligo has formed a double strand.

(2) Verification of the ability of the nanopolymer to load dsDNA: preparing polymer samples according to the N/P ratio (the molar ratio of nitrogen of the polymer to phosphorus in dsDNA) of 1, 2, 4, 6, 8, 10 and 20, after the gel is completely solidified, pulling out a comb, putting the gel into an electrophoresis tank, adding electrophoresis liquid to submerge the gel, adding 6X DNA loading into each sample, adding the sample into a sample loading hole after uniform mixing, carrying out electrophoresis at constant voltage of 100V for 40min, stopping an electrophoresis apparatus, taking out the gel, putting the gel into a gel imaging system, observing an electrophoresis strip under an ultraviolet lamp and taking a picture.

The results are shown in FIGS. 3 b-3 c, and it can be seen that when the N/P ratio is low, the polymer is not enough to completely encapsulate and complex the dsDNA, the free dsDNA moves to the anode to form a bright band, the band gradually weakens with the increase of the N/P ratio, and the N/P ratio is large enough to completely complex the dsDNA, and the band disappears; the results in the figure show that the dsDNA has good complexing effect when the N/P ratio is 10 and 20; when the N/P ratio is greater than 20, the dsDNA can be completely complexed.

Selecting the compound with the N/P ratio of 20 as a compound point in a cell experiment, and measuring the hydraulic diameter and the morphological structure of the compound, wherein the result is shown in a figure 3 a; as can be seen from the electron microscope result of FIG. 3a, the polymer nanoparticles are spherical, after aPD-L1 is loaded, obvious protein halos appear on the outer ring of the polymer structure, and the right side of FIG. 3a is the nanoparticle form under the environment with high content of MMP-2 matrix metalloenzyme around the simulated tumor.

Example 3 targeting of peritumoral cells to activate the Polymer cellular uptake status of the immunity

The dsDNA was fluorescently labeled with YOYO-1 to verify the uptake effect of antigen presenting cells. Using a six-well plate, selecting a polymer with the N/P ratio of 20 as a composite point in a cell experiment, wherein the group is Free DNA and Mannose-PEG_2k-Pasp (PEI) and Mannose + Mannose-PEG with competition by addition of excess Mannose_2k-Pasp (PEI), Raw264.7 cell pair uptake and intracellular distribution were observed by Leica SP8 confocal laser microscopy. The method comprises the following specific steps:

raw264.7 at 1X 10³Inoculating each cell/well into a 6-well plate (placing a clean glass slide in advance) to prepare a cell climbing sheet, and adding the group for incubation for 4 hours after the cells adhere to the wall; washing with fresh PBS twice, adding 4% paraformaldehyde to fix cells for 10min, adding 0.1% Triton X-100 to permeate cell membrane for 10min, washing with PBS twice, and dyeing nuclei with 1 μ g/mL Hochests for 5 min; and (3) sealing the chip by using an anti-fluorescence quenching sealing agent. The cell nucleus is excited by 405nm laser, and is detected by a DAPI fluorescence channel; YOYO-1 was excited with 491nm laser and detected by FITC fluorescence channel.

Referring to fig. 4, it can be seen that Free DNA group has no significant fluorescence signal, which indicates that DNA is not taken up, but vector group can effectively deliver dsDNA to cells, and we find that the amount of DNA taken up by cells is significantly reduced after competitive inhibition of mannose receptor on raw264.7 surface by mannose competitive inhibition group, so that the uptake of dsDNA is also enhanced by the presence of mannose in vector.

Example 4 cellular Western blot assay

Mouse bone marrow-derived dendritic cells (DC2.4) or mouse monocyte macrophage leukemia cells (Raw264.7) were plated at 1X 10 cells per well⁶Inoculating the cells in a 6-well plate at a density, wherein the drug groups are the nano polymer obtained in example 2 and the common STING agonist DMXAA, and after 2h, 6h, 12h and 24h after administration treatment, using RIPA lysis buffer containing protease inhibitor to lyse and extract total cell proteins; protein electrophoresis proteins of different molecular weights were separated using 12% gel, then transferred to PVDF membrane, which was blocked with 5% BSA solution for 1h, and then incubated with primary antibody STING, p-TBK1, p-IRF3 and β -Tublin overnight at 4 ℃; washing the membrane with TBST buffer solution for 3 times, then incubating with secondary antibody labeled with HRP for 1h, and washing the membrane with TBST for 3 times; finally, the corresponding immunoblot strip was exposed on a chemiluminescent detection system.

Results referring to fig. 5, the downstream signal angle compares the difference in STING activation by both: the nano polymer obtained in example 2 can still maintain phosphorylation signals (p-IRF3) within 24h, which shows that compared with the common STING agonist, the nano polymer has a more lasting effect of activating STING, and effectively solves the problem that general small molecule drugs are difficult to enter the cell interior through a cell membrane barrier to interact with STING, and are easily hydrolyzed by a large amount of enzymes in tissues to be inactivated.

Example 5 in vivo targeting experiments

The tumor cells of mouse breast cancer cells (4T1) are pre-planted into a BALb/c female white mouse, and the tumor volume reaches 150mm³Then, the nano-polymer obtained in example 2 was injected through the tail vein with cy 7.5-labeled Free aPD-L1; the distribution of cy7.5 in mice was photographed after anesthetizing the mice with isoflurane at a predetermined time.

Results referring to fig. 6, Free aPD-L1 can effectively target the tumor site in mice, demonstrating that it is desirable to target tumor tissue with aPD-L1, but takes longer and is degraded quickly; and the nano polymer obtained in example 2 has a short drug aggregation time and a longer action time due to the unique EPR effect of the nano particles, so that the curative effect of the drug is enhanced.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.